Summary HIV, like many other viruses (e.g. HTLV-1 and herpes viruses), disseminates from infected cells to new target cells much more efficiently by cell-to-cell transmission as compared to cell-free virion transmission. Cell- to-cell transmission occurs in an infectious or virological synapse (VS), where a tight cleft between an infected cell and a target cell is formed as a result of firmly adhering plasma membranes of the two apposing cells. VS formation is initiated upon HIV envelope gp120 interaction with CD4 which then recruits specific cell membrane proteins including the relevant co-receptor (CCR5 or CXCR4) and cellular adhesion molecules. Importantly, VS also requires active participation of intracellular signaling molecules in both the gp120-expressing donor cell and the CD4+ target cell. Our recent studies demonstrate that in the target T cell (efferent) side, a cascade of signaling events are triggered, starting from activation of CD4-associated Lck, to cause alteration of T cell motility and reorganization of actin cytoskeleton, each of which is potentially important for successful progression of the early stages of HIV transfer and infection at the VS. In the infected donor cell (afferent) side, virus assembly and budding are also facilitated by Lck, which binds and directs HIV gag to the plasma membrane in the infected T cell. The importance of signaling molecules in VS formation presents an opportunity to exploit these signal transduction pathways for blocking HIV transmission and infection. In this application we will focus on HIV transmission from infected CD4 T cells to target CD4 T cells, the most common cell type infected by HIV, and define the membrane-proximal signaling molecules and pathways activated in the infected donor and uninfected target cells that are required for VS formation. Identifying these molecules and pathways will allow us the opportunity to inhibit their activity to stop HIV transmission through the VS. To achieve this, we will evaluate the importance of Lck activation and its downstream signaling events for efficient VS formation and cell-cell transmission of HIV (Aim 1). We will determine membrane-proximal signaling molecules that are recruited to VS in the infected donor cells and compare them with those found in the target T cells (Aim 2). The involvement of Lck and other activation signals in cytoskeleton reorganization that facilitates VS-mediated virus egress and entry will also be studied (Aim 3). Finally, we will determine how HIV Env interaction with the 1427 integrin on the target CD4 T cells affects HIV transmission via the VS, and VS formation and signaling (Aim 4). Identification of signaling molecules and pathways essential for VS formation should pave the way for the development of signal transduction inhibitors as a novel class of HIV inhibitors. There is a growing interest for such anti-HIV modalities as they circumvent the unprecedented problem of HIV-1 genetic and antigenic variability, which has hampered the development of efficacious HIV/AIDS vaccines and has led to the emergence of drug-resistant viruses.